Dynamic light scattering (DLS) is a well established technique for measuring particle size over the size range of a few nanometers to a few microns. DLS determines particle size from the analysis of the Brownian motion of suspended particles. Light scattered from a moving particle has a Doppler light frequency shift imparted to it. Scattering from a group of particles will have a distribution of shifts from the randomly moving particles. Measuring the Doppler shifts therefore provides a means of measuring the Brownian motion of the particles and hence provides a means of determining the size of the particles.
Most fine particle size analyzers determine particle size by measuring the Doppler shift of light as it is scattered by moving particles. Smaller particles move faster, causing a greater Doppler shift in the light they scatter. With conventional analyzers, light passes completely through an extremely dilute suspension and scatters in all directions. The detector measuring the Doppler shifts of the scattered light has no high-level signal to reference against. The resulting low-level signals require amplification from photomultipliers, which can introduce noise errors.
In the Microtrac.RTM. ultrafine particle analyzer (sold by Leeds & Northrup, a unit of General Signal Corporation), light travels to the sample via an optical wave guide. A mirror reflects some of the light, creating a high-level reference signal. Moving particles back-scatter the light penetrating the mirror. The Microtrac.RTM. ultrafine particle analyzer combines the reflected and back-scattered light to create a high-level signal strong enough to be fed directly to a reliable solid-state photodetector.
The Microtrac.RTM. ultrafine particle analyzer is typically operated such that a stream of liquid carrier containing the particles to be analyzed are sent past a photoprobe (e.g., an optical probe). If the solution containing the particles to be analyzed did not produce the desired result, other solutions would be produced and sent past the photoprobe until an acceptable result is obtained. This procedure is acceptable for solutions which are inexpensive and readily available. However, in the case where the solutions to be passed by the photoprobe are relatively expensive, it would not be desirable to continuously send new solutions into the sample cell until the desired results are obtained. This is particularly true in the case of proteins wherein the high cost of the material substantially outweighs the information obtained by conducting dynamic light scattering analysis of the particles contained therein. Accordingly, biochemical researchers have foregone the DLS analysis of proteins and other expensive particles due to their high cost and the need by conventional DLS analyzers to use substantial volumes of solution containing such particles in order to arrive at acceptable results.
It would be most desirable to be able to perform DLS analysis of proteins and other expensive materials, without using large sample volumes. The present inventors have developed a unique DLS analyzer which uses a small volume sample for measurement of particle size, while allowing for the in-situ adjustment of the concentration of each sample using inexpensive continuous flow fluids, such as inhibitors, substrates, salts, etc. As such, the concentration of the sample can be gradually changed while it is continuously analyzed, without having to continuously replace the sample volume if the results obtained are not acceptable.
Several very important areas of protein research can benefit from this DLS analysis having continuous flow dialysis. Prior to the present invention, DLS technology had been primarily applied only to "model" proteins which are available in huge quantities and at low cost in order to prove its benefits to protein research. The present invention allows the use of DLS technology in three major areas of macromolecular research which would not otherwise be able to take advantage of this kind of analysis, i.e., protein subunit oligomerization, enzyme/enzyme interactions, and protein aggregation/crystallization.
Without a continuous flow set-up such as the present invention, DLS technology is too sample costly to be routinely used in protein research. DLS systems presently available require too large of a sample volume, too high sample concentration, and multiple sample handlings for each data point collected. Also, in order to collect multiple data points, large amounts of research time are required. The continuous flow dialysis approach of the present invention would require only one sample handling step and would save research time by allowing the computer to automatically change sample components and collect the necessary data in a single or multidimensional manner.
The ability to adjust and alter the liquid carrier in the cell chamber has several advantages: lower sample loss, less sample preparation time, virtually continuous sample monitoring, and automated analysis.
The present invention also provides many additional advantages which shall become apparent as described below.